POS0250 PLASMA MITOCHONDRIAL DNA AS A BIOMARKER IN THE DIAGNOSIS AND FOLLOW-UP OF ANCA-ASSOCIATED VASCULITIDES
Authors: S. Giaglis, D. Kyburz, J. Thiel, N. Venhoff, U. Walker
Journal: Annals of the Rheumatic Diseases (2022)

Background Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) include granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) (1, 2). ANCA recognize the antimicrobial proteins proteinase 3 (PR3) or myeloperoxidase (MPO) (1,2) and trigger the formation of neutrophil extracellular traps (NETs), which release DNA into the extracellular space and systemic circulation. This cell-free (cf)DNA induces endothelial damage, vascular inflammation and necrosis (3). Objectives The nature, diagnostic and prognostic value of cfDNA in AAV is still unknown. The aim of the present study was to examine the clinical utility of cfDNA quantification as a biomarker in AAV. Methods Total DNA was isolated from platelet-free plasma samples of healthy controls (HC) and consecutive AAV patients. Plasma and clinical data were collected at baseline and follow-up. Copy numbers were quantified by qPCR for mtDNA (ATP-6 gene) and nuclear (n) DNA (GAPDH gene) (4). Patients with eosinophilic GPA (EGPA) were excluded. Results Ninety-two HC (median age 51 ± 9, 48.2% female) and 104 AAV patients (median age 64 ± 10, 48% female, mean BVAS: 0; range: 0-40) were available for analysis. Eighty-four (80.8%) of these patients were diagnosed with GPA, and 20 with MPA (19.2%). mtDNA levels were significantly elevated in AAV plasma (8.7x10 7 copies/ml plasma, 95% CI: 5.3x10 7 to 1.3 x10 8) ), compared to HC plasma (6.7x10 6 copies/ml plasma, 95% CI: 5.4x10 6 to 9.1x10 7 , p&lt;0.0001). nDNA levels in contrast did not differ between AAV (4.0x10 6 copies/ml plasma, 95%CI: 2.7x10 6 to 5.0x10 6 ) and HC (3.3x10 6 copies/ml plasma, 95%CI: 2.4x10 6 to 4.7x10 6 , p=0.30). ROC analysis showed that a cut-off value of 1.3x10 7 mtDNA copy numbers differentiated between AAV and HC with 89.4% sensitivity, 82.6% specificity and an AUC of 0.94. For AAV patients with active AAV, a cut-off value of 2.9x10 7 mtDNA copy numbers differentiated between AAV and HC with 96.1% sensitivity, 98.9% specificity and an AUC of 0.99 (Figure 1a). Figure 1. ( a. ) ROC curve for mtDNA plasma concentrations to discriminate between HC and active AAV patients (BVAS&gt;0). AUC: area under the curve. ( b. ) Plasma mtDNA levels distinguish between AAV patients with active disease versus patients in a state of remission. Whiskers represent 95% CI. ( c. ) Plasma mtDNA levels in AAV patients correlate with the evolution of disease activity at follow-up. With the exception of the peripheral nervous system involvement, there was no association of mtDNA elevation with any particular type of active organ involvement at the time of blood sampling. A positive correlation between all cell-free DNA species and anti-MPO antibody titres was observed, as expected (for cfDNA, nDNA and mtDNA - r=0.25, p=0.01; r=0.21, p=0.02; r=0.22, p=0.02, respectively). AAV patients with active disease (BVAS&gt;0) had a mean of 2.0x10 8 copies/ml of mtDNA in plasma which was higher compared to HC (p&lt;0.0001) and AAV patients in remission (BVAS=0) (6.2x10 7 copies/ml, p=0.03). For nDNA on the other hand, there were similar levels in active disease as in remission (5.3x10 6 and 4.8x10 6 copies/ml, respectively; p=0.64) (Figure 1b). Follow-up data were available for 27 AAV patients (median follow-up: 6 ± 6 months, IQR: 12). Longitudinal changes in mtDNA levels robustly correlated with changes in BVAS (r=0.56, p=0.002, Figure 1c). Conclusion The quantification of cell free mtDNA - but not nDNA - copy numbers allows a sensitive and specific distinction between healthy individuals and patients with active AAV. mtDNA levels correlate cross sectionally with disease activity in AAV patients. Plasma mtDNA quantification may therefore aid in the diagnosis of AAV and in monitoring AAV activity. References [1]Kitching, A.R., et al. Nat Rev Dis Primers 6, 71 (2020). [2]Kallenberg, C. Nat Rev Rheumatol 10, 484–493 (2014). [3]Kessenbrock, K., et al. Nat Med 15, 623–625 (2009). [4]Giaglis S, et al. RMD Open 2021; 7: e002010.(2021) Disclosure of Interests None declared